苯丙氨酸CoA酰基转移酶原核表达系统的构建和表达

目的:获得大量的重组红豆杉苯丙基转移酶(BAPT)，为紫杉醇半合成代谢提供廉价的催化剂。方法：根据DNA重组技术，构建原核表达载体pET-BAFF，使目的基因位于原核T7启动子下游，IPTG诱导基因表达。结果：BAPT高效表达，重组蛋白主要以包涵体的形式存在。结论：为获得可溶性重组蛋白BAFF奠定了理论基础。     

乳糖作为诱导剂对重组目的蛋白表达的影响

将重组粒细胞-巨噬细胞集落刺激因子/白细胞介素3(GM-CSF/IL-3)融合蛋白表达菌BL21(DE3)(pFu)作为研究对象,对于以乳糖作为诱导剂时重组目的产物的诱导表达规律进行了深入的研究。分析比较了不同培养基中,不同生长阶段进行诱导对于产物表达的影响。对诱导所需的乳糖浓度、诱导持续时间长短等因素亦进行了研究。实验结果表明,在对诱导条件进行优化控制的前提下,利用乳糖作为诱导剂可以达到与IPTG类似的诱导效果。随后的研究中,将乳糖作为诱导剂应用于高密度发酵过程。这些研究结果为乳糖作为诱导剂最终应用于重组基因工程药物的工业化生产提供了有益的参考和借鉴。     

VEGF121-KLAK融合蛋白在大肠杆菌BL21(DE3)中的表达及纯化

目的:在大肠杆菌中高效表达并纯化VEGF121与两性分子KLAK的融合蛋白,为进一步研究其抗肿瘤血管形成作用奠定基础。方法:用RT-PCR法扩增目的基因,插入表达载体pET28a后,转化大肠杆菌BL21(DE3),经IPTG诱导表达重组蛋白,对产物进行SDS-PAGE及Western印迹分析。结果:克隆出目的基因,构建了融合蛋白表达载体,诱导表达后经SDS-PAGE检测表明获得了目的条带。结论:在大肠杆菌中高效表达并纯化了融合蛋白VEGF121-KLAK。     

人巨细胞病毒编码蛋白pUL23在E.coli BL21(DE3)中的表达和纯化

大肠杆菌中高效表达携带组氨酸标签的人巨细胞病毒皮层蛋白pUL23,并进行纯化以及鉴定.提取感染HCMV Towne病毒株的HFF细胞的总RNA,逆转录为cDNA作为模板,经PCR获得UL23的基因片段,将此片段插入表达载体pET-28a(+),构建pET28a(+)-UL23重组质粒.将pET28a(+)-UL23转化至大肠杆菌BL21( DE3),进行IPTG诱导表达.表达产物经Western blotting分析后进行发酵,再用Ni sepharose亲和层析纯化,纯化产物进行SDS-PAGE和Western blotting检测.结果表明,成功构建pET28a(+)-UL23原核表达载体,表达及纯化了His-pUL23融合蛋白.为进一步研究pUL23奠定基础.     

大肠杆菌BL21(DE3)lpxM突变株的构建

目的:lpxM基因失活可以产生极低内毒素活性的脂多糖。构建大肠杆菌BL21(DE3)的lpxM突变株,并考察该突变株的生长状态和表达重组蛋白的能力。方法:构建同源臂长500bp左右的打靶载体,借助Red同源重组系统,使E.coliBL21(DE3)的lpxM基因发生插入失活,再导入编码FLP位点特异性重组酶的质粒pCP20去除抗性基因。PCR鉴定发生插入突变的菌株,应用SDS-PAGE分析突变前后的脂多糖,考查对重组蛋白表达的影响。结果:PCR鉴定结果说明lpxM基因发生了插入突变。与出发株比较,突变株的脂多糖电泳图谱发生了明显变化,但其生长状态与表达重组蛋白的能力与出发株基本一致。结论:大肠杆菌BL21(DE3)的lpxM突变株可以用于重组蛋白的表达。     

TAT-hEGF融合蛋白在E.coli BL21(DE3)中高效自我表达

为了获得TAT-hEGF融合蛋白在E.coliBL21(DE3)中高效表达,构建了原核表达载体pRSET-tat-hegf,将其转化E.coliBL21(DE3)得到重组工程菌BL21(DE3)/pRSET-tat-hegf。工程菌在无IPTG的诱导下实现了高效表达,TAT-hEGF融合蛋白的表达量占总菌体蛋白的45.6%,主要以包涵体形式存在。     

Production of Recombinant Proteins in the lon-Deficient BL21(DE3) Strain of Escherichia coli in the Absence of the DnaK Chaperone

To eliminate unavoidable contamination of purified recombinant proteins by DnaK, we present a unique approach employing a BL21(DE3) ΔdnaK strain of Escherichia coli. Selected representative purified proteins remained soluble, correctly assembled, and active. This finding establishes DnaK dispensability for protein production in BL21(DE3), which is void of Lon protease, key to eliminating unfolded proteins.Obtaining substantial amounts of pure protein is essential in innumerable biological studies and indispensable to the biochemical characterization of proteins. The ease of growth, well-characterized genetics, and the large number of tools for gene expression have long made Escherichia coli the organism of choice for protein overproduction. The BL21(DE3) strain is widely used for recombinant protein production because of its engineered capacity to produce T7 polymerase and its deficiency in Lon and OmpT proteases.DnaK is an abundant protein (about 1% of the total protein of E. coli) (17) that interacts with a wide range of newly synthesized polypeptides (28) and assists their proper folding and assembly into oligomers by preventing protein aggregation. DnaK, together with ClpB ATPase, is also required to disaggregate preformed protein aggregates (12, 20), and it participates in the degradation of damaged proteins by Lon and ClpP (26, 27).The inactivation of dnaK has been shown previously to increase the insoluble fractions of certain aggregation-prone recombinant proteins (7), and DnaK alleviates the aggregation of certain heterologous proteins when coproduced with the protein of interest (8). However, fruitful coproduction of recombinant proteins with chaperones has been challenged by recent findings demonstrating that chaperones increase the solubility but not necessarily the quality of proteins (13, 15).The DnaK binding site, a five-residue hydrophobic core flanked by two basic residue-enriched regions, occurs on average every 36 residues in protein sequences (25). One consequence is unwanted DnaK contamination of recombinant proteins during purification in E. coli, even after several chromatographic steps (1, 3, 11, 14, 16, 21, 23).One challenge in protein purification is to obtain the highest level of purity in the fewest steps. Biologically active impurities can jeopardize research or therapeutic applications even if present in trace amounts. One approach developed to circumvent DnaK contamination is extensive washing of columns with ATP since DnaK in its ATP-bound state has low affinity for protein (3). However, this strategy lengthens the purification procedure, is expensive, and is of inconsistent effectiveness (1, 14).In an attempt to eliminate DnaK contamination, we have investigated whether recombinant proteins could be produced in the absence of DnaK. Toward that end, we constructed a ΔdnaK derivative of the extensively employed E. coli B host strain BL21(DE3). The consequences of the absence of DnaK for the production, solubilities, correct assembly, and activities of several recombinant proteins in BL21(DE3) have been studied. Obtaining a BL21(DE3) ΔdnaK strain has allowed us to elucidate to what extent such a major E. coli chaperone is indispensable to protein overproduction in the particular genetic context of an E. coli strain that lacks Lon, an ATP-dependent protease responsible for degrading unfolded proteins (10).dnaK in BL21(DE3) was inactivated by the introduction of a null allele, ΔdnaK::Kan, from the E. coli PopC4617 strain by P1 transduction (see Table S1 in the supplemental material). Transductants were selected at 30°C in Luria-Bertani medium complemented with kanamycin. The absence of dnaK was verified by colony PCR using the specific primers dnaK-Nter (5′-GGTAAAATAATTGGTATCGACCTGG-3′) and dnaK-Cter (5′-GTCTTTGACTTCTTCAAATTCAGCG-3′) (see Fig. S1 in the supplemental material). Immunoblotting using an anti-DnaK antibody showed that the obtained transductant (EN2) did not produce DnaK (see Fig. S1 in the supplemental material). The EN2 strain has been deposited at the Collection Nationale de Culture de Microorganismes at the Institut Pasteur (with identification number CNCM I-3863).E. coli K-12 dnaK mutants usually have a narrow range of permissive temperatures for growth (around 30°C) and exhibit multiple cellular defects, such as impaired cell division and the inhibition of DNA and RNA synthesis (4, 6, 18, 22). Inactivating dnaK in the genetic background of BL21(DE3), an E. coli B strain which is already deficient in OmpT and Lon proteases, did not lead to a dramatic difference in the exponential growth rate at 30°C, but at stationary phase, EN2 cells exhibited slightly reduced ability to form colonies on plates (data not shown). As expected for dnaK mutants, EN2 cells demonstrated impaired growth at 42°C (data not shown). Inactivating dnaK in BL21(DE3) did not induce major morphological defects, and EN2 cells were never found to form long filaments, as dnaK mutants with other genetic backgrounds have previously been reported to do (5) (data not shown). Therefore, the EN2 strain can easily be cultivated at 30°C.We next investigated whether inactivating dnaK in BL21(DE3) would impair the production and solubilities of different recombinant proteins (whose features are summarized in Table S2 in the supplemental material). These proteins belong to organisms of different kingdoms, and their molecular masses range from 19 to 51 kDa; therefore, they potentially correspond to DnaK substrates since the masses of polypeptides interacting with DnaK range from 14 to 90 kDa (28). Many of them exist as oligomers and may require the assistance of DnaK for proper assembly. These proteins were also chosen for their different levels of production and solubility in E. coli. Four of them (CpxP, ClpP1, PA28α, and proteasome-activating nucleotidase [PAN]) are totally soluble, and two of them (ClpP2 and green fluorescent protein [GFP]) are aggregation prone and may require the presence of DnaK to prevent their aggregation. Importantly, all these proteins were contaminated by DnaK when purified from E. coli (see Fig. ​Fig.33).Open in a separate windowFIG. 3.Purification of recombinant proteins in the absence of DnaK. Aliquots of 10 μg of CpxP, ClpP1, ClpP2, GFP, and PAN purified from BL21(DE3) cells (lanes 1, 3, 5, 7, and 9) or EN2 cells (lanes 2, 4, 6, 8, and 10) were loaded onto an SDS-12% PAGE gel. (A) Proteins were revealed by Coomassie blue staining. (B and C) DnaK (B) and GroEL (C) were detected by Western blotting. Sizes of molecular mass markers (lanes MW) are given in kilodaltons and indicated to the left of the gel. The asterisk indicates the position of DnaK on the gel. The two major bands in the purified PAN sample correspond to full-length 50-kDa His-PAN and the 40-kDa PAN fragment resulting from the internal initiation of translation, which copurify as oligomeric complexes (30). The gels shown are representative of results from at least three independent experiments.The production of recombinant proteins in exponentially growing BL21(DE3) and EN2 cells in Luria-Bertani medium at 30°C was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 2 h. The same biomasses of BL21(DE3) and EN2 cells were sonicated in 1 ml of lysis buffer (50 mM Tris, pH 7.5, 100 mM KCl, 1 mM dithiothreitol). Soluble proteins were separated from aggregated proteins and cellular debris by 30 min of centrifugation at 14,000 × g and 4°C. Pellets containing protein aggregates were resuspended in 1 ml of Tris, pH 7.5, containing 1% sodium dodecyl sulfate (SDS). Total extracts and soluble and insoluble fractions were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Levels of production and solubility of recombinant proteins in the absence of DnaK. Aliquots of 10 μg of total extracts (T-un and T) and soluble (S) and insoluble (P) fractions from uninduced (T-un) and IPTG-induced (T, S, and P) BL21(DE3) and EN2 cells overexpressing CpxP (A), ClpP1 (B), PA28α (C), ClpP2 (D), GFP (E), or PAN (F) were analyzed by SDS-12% PAGE on gels stained by Coomassie blue. Sizes of molecular mass markers (lanes MW) are given in kilodaltons and indicated to the left of each gel. Arrowheads indicate the positions of recombinant proteins. The gels shown are representative of results from at least three independent experiments.The levels of production of all tested proteins in EN2 and BL21(DE3) cells were similar, as demonstrated by the protein amounts in total extracts (Fig. ​(Fig.1,1, lanes T). Moreover, dnaK inactivation did not affect the solubilities of recombinant proteins, even those such as CpxP (Fig. ​(Fig.1A),1A), ClpP1 (Fig. ​(Fig.1B),1B), and PA28α (Fig. ​(Fig.1C)1C) produced in high amounts or those such as ClpP2 (Fig. ​(Fig.1D)1D) and GFP (Fig. ​(Fig.1E)1E) prone to aggregation. These findings were surprising since the function of the DnaK chaperone is to prevent protein aggregation during synthesis and to cooperate with DnaJ, GrpE, and ClpB in the disaggregation of aggregates. It seems that, even for aggregation-prone recombinant proteins, solubility may not necessarily be dependent on endogenous DnaK. This finding may reflect the different folding requirements of specific proteins. Another explanation may be the presence of another chaperone with an overlapping conjoint function. In fact, a consequence of the absence of DnaK in cells is higher levels of production of heat shock proteins such as GroEL/GroES (29). Consistent with these data, EN2 cells produced higher amounts of GroEL than BL21(DE3) cells (data not shown), and these higher amounts may compensate for the absence of DnaK in preventing protein aggregation, as was shown previously for endogenous E. coli proteins and other recombinant proteins (8, 28). An abundance of different chaperones playing nonspecialized roles in recombinant protein folding in E. coli cells may permit toleration of the loss of DnaK, without impairing cell capacity as a protein production factory.Since the examined proteins could fold and assemble independently of DnaK, we next tested whether a protein known to interact with DnaK could be produced in the absence of this chaperone. Nemo, the IκB kinase complex regulatory component of the NF-κB signaling pathway in eukaryotes, was shown previously to tightly bind and be contaminated by DnaK when produced in E. coli (1). When recombinant His-tagged Nemo was produced in BL21(DE3) under our conditions, it was barely detectable on electrophoresis gel (Fig. 2A and B). However, immunodetection using an anti-His₆ antibody (Roche) at a 1:2,000 dilution showed that the absence of DnaK resulted in an increase in Nemo production (Fig. ​(Fig.2D).2D). When Nemo was produced in higher amounts, most of the protein was found in the soluble fraction, indicating that it could be produced as a soluble species in the absence of DnaK (Fig. ​(Fig.2D,2D, lane 5). Increased production of Nemo in the absence of DnaK could be explained by a role of this chaperone in Nemo degradation. Producing Nemo in a BL21(DE3) strain that is deficient in the protease ClpP did not increase its cellular amount (data not shown), indicating that if Nemo was degraded in a DnaK-dependent manner in BL21(DE3) (which already lacks Lon protease), ClpP was not responsible for this proteolysis or the absence of ClpP was compensated for by another protease.Open in a separate windowFIG. 2.Levels of production and solubility of recombinant Nemo in the absence of DnaK. Aliquots of 10 μg (A and C) or 20 μg (B and D) of total extracts (T) and soluble (S) and insoluble (P) fractions from uninduced and IPTG-induced BL21(DE3) and EN2 cells overexpressing Nemo were loaded onto an SDS-10% PAGE gel. Proteins were detected by Coomassie blue staining (A and C), and His-tagged Nemo was detected by Western blotting (B and D). Sizes of molecular mass markers (lanes MW) are given in kilodaltons and indicated to the left of each gel. The gels shown are representative of results from at least three independent experiments.We next tested whether recombinant proteins produced in the absence of DnaK would remain soluble and active during their purification. Samples of 200 ml of cells overproducing CpxP, ClpP1, ClpP2, or GFP or 500 ml of PAN-overproducing cells were sonicated in 2 ml of lysis buffer (50 mM NaH₂PO₄, pH 8.0, 300 mM NaCl, 10 mM imidazole). The soluble fraction obtained after 30 min of centrifugation at 38,000 × g and 4°C was loaded onto 400 μl of nickel-nitrilotriacetic acid resin, and His-tagged proteins were purified according to the recommendations of the resin manufacturer (Qiagen). After elution, His-tagged proteins were dialyzed against 50 mM Tris, pH 7.5, concentrated, and analyzed by electrophoresis.By this procedure, recombinant proteins were purified to the levels of homogeneity indicated in Fig. ​Fig.3A.3A. Samples of 10 μg of purified proteins were used for the immunodetection of contamination by DnaK (using an anti-DnaK antibody from Stressgen at a 1:2,000 dilution). We found that DnaK in BL21(DE3) cells contaminated all preparations of purified recombinant proteins, albeit to different extents (Fig. ​(Fig.3B,3B, lanes 1, 3, 5, 7, and 9). As expected, dnaK inactivation prevented such contamination (Fig. ​(Fig.3B,3B, lanes 2, 4, 6, 8, and 10). It is noteworthy that most of the proteins purified from BL21(DE3) were also contaminated by GroEL, although this contamination was minor. In EN2 cells, where GroEL expression is increased, we did not systematically observe greater contamination by GroEL (Fig. ​(Fig.3C).3C). Moreover, CpxP and GFP, the proteins that exhibited the greatest DnaK contamination, were not the most contaminated by GroEL, and GroEL did not copurify with PAN in the absence of DnaK. Thus, the absence of DnaK did not necessarily lead to a higher level of contamination by GroEL.Despite the absence of DnaK, all purified recombinant proteins remained soluble even after being concentrated. Since some aggregates are soluble and solubility does not always guarrantee a native active conformation (15, 19), the activities (when readily measurable) or native conformations of some of the purified proteins were examined. One microgram of purified PAN was used to measure ATP hydrolysis at 55°C as described earlier (2). PAN proteins purified from BL21(DE3) and EN2 cells had comparable ATPase activities, with means ± standard errors of 762.33 ± 145.51 and 968.32 ± 198.85 nmol mg⁻¹ h⁻¹ (n = 3), respectively. The fluorescence emission spectrum (at an excitation wavelength of 400 nm) of GFP purified from EN2 cells was indistinguishable from that of GFP purified from BL21(DE3) cells (Fig. ​(Fig.4A),4A), indicating that GFP remained correctly folded when produced in the absence of DnaK. CpxP, a component of the Cpx signal transduction pathway, was the protein that exhibited the greatest DnaK contamination (11). It self-associates into dimers (M. Miot and J.-M. Betton, unpublished data), and to test its correct assembly, 100 μl of purified CpxP at 1 mg/ml in a buffer of 25 mM Tris, pH 7.5, and 150 mM NaCl was loaded onto a size exclusion chromatography column (Superdex 200 HR10/30; GE Healthcare) and eluted with the same buffer at a flow rate of 0.5 ml/min. Recombinant CpxP purified from BL21(DE3) eluted at a volume of 14.98 ml (Fig. ​(Fig.4B),4B), corresponding to a species with an apparent molecular mass of 39.85 kDa (a dimer of Cpx). Recombinant CpxP purified from EN2 eluted at a nearly identical volume of 14.97 ml (Fig. ​(Fig.4B).4B). Thus, the absence of DnaK did not alter CpxP dimeric assembly and did not produce any soluble higher-molecular-mass aggregate species.Open in a separate windowFIG. 4.Folding and assembly of proteins in the absence of DnaK. (A) Fluorescence emission spectra of 8-μg/ml GFP preparations purified from BL21(DE3) and EN2 cells, recorded with an FP-6200 spectrofluorimeter (Jasco) at a scan rate of 250 nm min⁻¹ using a bandwidth of 5 nm for both excitation and emission beams. The spectra shown are representative of results from at least two independent experiments. (B) Size exclusion chromatograms for CpxP proteins purified from BL21(DE3) and EN2 cells. Arrowheads indicate the elution volumes of the standards, and their masses are given in kilodaltons. The chromatograms shown are representative of results from at least three independent experiments.Altogether, these findings indicate that high levels of correctly folded, assembled, and active soluble recombinant proteins can be produced in the absence of endogenous DnaK chaperone in BL21(DE3). Surprisingly, our study showed that the inactivation of dnaK in BL21(DE3), which does not contain Lon, did not result in an increase in the aggregation of recombinant proteins, as was seen previously in E. coli K-12 (24). It seems that in BL21(DE3) cells, and in E. coli B cells in general, factors other than DnaK and Lon may be fundamental in managing the accumulation of aggregated proteins. Through the detailed characterization of a BL21(DE3) ΔdnaK strain and testing of the production of proteins of different natures, origins, and sizes, including aggregation-prone proteins, our study demonstrates that this EN2 strain offers a strategy that can be generally and extensively used to avoid unwanted contamination by DnaK. In addition, since DnaK has ATPase activity, the EN2 strain is particularly well suited for the production and purification of recombinant ATPases, eliminating the undifferentiable ATPase contamination. Given that GroEL, another major chaperone in E. coli, has also been found to contaminate purified recombinant proteins (9), it would be of additional interest to find conditions under which both dnaK and groEL could be eliminated in the BL21(DE3) strain without impairing its survival and its remarkable protein factory capacities.     

Recombinant production of active Streptococcus pneumoniae CysE in E. coli facilitated by codon optimized BL21(DE3)-RIL and detergent

Abstract

The emergence of drug resistance in Streptococcus pneumoniae (Spn) is a global health threat and necessitates discovery of novel therapeutics. The serine acetyltransferase (also known as CysE) is an enzyme of cysteine biosynthesis pathway and is reported to be essential for the survival of several pathogenic bacteria. Therefore, it appears to be a very attractive target for structure–function understanding and inhibitor design. This study describes the molecular cloning of cysE from Spn in the pET21c vector and efforts carried out for expression and purification of active recombinant CysE. Significant expression of recombinant Spn cysE could be achieved in codon optimized BL21(DE3)-RIL strain as opposed to conventional BL21(DE3) strain. Analysis of codon adaptation index (CAI) with levels of eukaryotic genes and prokaryotic cysEs expressed in heterologous E. coli host suggests that codon optimized E. coli BL21(DE3)-RIL may be a better host for expressing genes with low CAI. Here, an efficient protocol has been developed for recovery of recombinant Spn CysE in soluble and biologically active form by the usage of nonionic detergent Triton X-100 at a concentration as low as 1%. Altogether, this study reports a simple strategy for producing functionally active Spn CysE in E. coli.     

Overexpression of a lethal methylase,M.TneDI,in E. coli BL21(DE3)

A pET-based vector pDH21 expressing the methylase, M.TneDI (recognizing CGCG) from Thermotoga was constructed, and transformed into E. coli BL21(DE3). Despite E. coli BL21(DE3) being McrBC positive, 30 transformants were isolated, which were suspected to be McrBC^? mutants. The overexpression of M.TneDI was verified by SDS-PAGE analysis. Compared to the previously constructed pJC340 vector, a pACYC184 derivative expressing M.TneDI from a tet promotor, the newly constructed pDH21 vector improved the expression of the methylase about fourfold, allowing complete protection of DNA substrates. This study not only demonstrates a practical approach to overexpressing potential lethal proteins in E. coli but also delivers a production strain of M.TneDI that may be useful in various in vitro methylation applications.     

基因重组大肠杆菌表达HrpNEcc蛋白的发酵条件及诱导条件优化

对已构建好的表达HrpNEcc蛋白的工程菌BL21(DE3)/pET30a(+)hrpN Ecc的摇瓶发酵条件及乳糖诱导进行优化, 通过在7L发酵罐中放大发酵实验,以期提高蛋白产量并降低生产成本。在摇瓶中优化的发酵及诱导条件是：5% 的接种量,TB培养基,菌体培养至对数生长前期,添加3g/L外源诱导剂乳糖时,HrpNEcc蛋白产量可达417.60mg/L,比不添加乳糖时提高了36.73%,比用IPTG诱导时提高了16.85%。7L发酵罐中发酵,获得菌体湿重达到57.24g/L（WCW）,可溶性HrpNEcc蛋白产量占细胞总蛋白的50.2%,为3.29 g/L。     

鼠疫菌V抗原基因在大肠杆菌中的克隆及表达

从鼠疫杆菌野毒株总DNA中利用PCR方法扩增出V抗原编码基因,将此基因克隆到大肠杆菌的表达质粒pET42b(+)中,经IPTG诱导后,在大肠杆菌BL21(DE3)细胞中成功地表达出鼠疫菌V抗原蛋白。目的蛋白表达量占菌体总蛋白的32.8%。     

Expression and characterization of soybean seed coat peroxidase in Escherichia coli BL21(DE3)

Soybean seed coat peroxidase (SBP) is a valuable enzyme having a broad variety of applications in analytical chemistry, biochemistry, and food processing. In the present study, the sscp gene (Gene ID: 548068) was optimized based on the preferred codon usage of Escherichia coli, synthesized, and expressed in E. coli BL21(DE3). SDS-PAGE and western blot analysis of this expressed protein revealed that its molecular weight is approximately 39?kDa. The effects of induction temperature, concentration of isopropyl-β-D-thiogalactoside and hemin, induction time, expression time were optimized to enhance SBP production with a maximum activity of 11.23?U/mL (8.64?U/mg total protein). Furthermore, the kinetics of enzyme-catalyzed reactions of recombinant protein was determined. When 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was used as substrate, optimum reaction temperature and pH of the enzyme were 85°C and 5.0, respectively. The effects of metal ions on the enzymatic reaction were also further investigated. The SBP was successfully expressed in E. coli BL21(DE3) which would provide a more efficient production strategy for industrial applications of SBP.     

乳糖诱导重组尿酸酶基因在大肠杆菌中的表达

对用乳糖替代异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组产朊假丝酵母尿酸酶基因在E.coli JM109(DE3)中表达进行了研究,拟建立一种高效低成本的生产重组尿酸酶的工艺路线。通过摇瓶试验对诱导所采用的乳糖浓度,诱导时机和诱导持续时间进行了优化,并考察在乳糖诱导下的目的产物表达动力学,随后在5 L发酵罐上进行扩大化培养以验证摇瓶优化的结果,进一步将乳糖作为诱导剂应用于高密度发酵过程。实验结果表明乳糖诱导的最佳浓度为5 g/L,最佳诱导时机是对数生长期中后期,诱导持续时间为9~10h;按照优化的条件在摇瓶和5 L发酵罐上进行分批培养,重组尿酸酶最大表达量可达菌体总蛋白的26%左右,可溶性蛋白的36%左右,略高于IPTG的诱导效果;高密度发酵过程菌体终密度达到OD600值40以上,尿酸酶表达量占菌体总蛋白25%左右。     

金属硫蛋白与单抗SZ51重组蛋白在大肠杆菌菌株BL21(DE3)pLysS中表达的研究

从基因工程水平构建了小鼠金属硫蛋白与抗人活化血小板单抗ＳＺ５１单链抗体的重组基因产物,并在大肠杆菌菌株ＢＬ２１（ＤＥ３）ｐＬｙｓＳ中成功地进行了表达．该重组蛋白以包涵体形式存在于菌体蛋白中,分子量为３８ｋＤ,ＥＬＩＳＡ实验证明该重组蛋白既具有血栓部位活化血小板的单抗活性,又具有小鼠ＭＴ单抗的活性     

Enhancement of Recombinant Hemoglobin Production in Escherichia coli BL21(DE3) Containing the Plesiomonas shigelloides Heme Transport System

To produce recombinant hemoglobin in Escherichia coli, sufficient intracellular heme must be present, or the protein folds improperly and is degraded. In this study, coexpression of human hemoglobin genes and Plesiomonas shigelloides heme transport genes enhanced recombinant hemoglobin production in E. coli BL21(DE3) grown in medium containing heme.     

破伤风毒素C片段(TTc)在大肠杆菌中的表达、优化与鉴定

HTSS以一株破伤风生产菌株基因组DNA为模板,通过上游引物中几个碱基的修改,PCR扩增出破伤风毒素C片段(TTc)基因,构建了原核表达质粒pET-42(b)/TTc,在大肠杆菌BL21(DE3)中表达。重组蛋白分子量约50kD,表达量为22%,超声波破碎显示为可溶性重组蛋白。通过对培养基、诱导时间、诱导温度的优化,重组蛋白的表达量和可溶性均有提高。Western blotting检测表达产物可与破伤风C片段单克隆抗体产生特异的免疫反应。该工作为亚单位疫苗或载体蛋白的开发奠定了基础。     

Rapid label‐free quantitative analysis of the E. coli BL21(DE3) inner membrane proteome

Biological membranes define cells and cellular compartments and are essential in regulating bidirectional flow of chemicals and signals. Characterizing their protein content therefore is required to determine their function, nevertheless, the comprehensive determination of membrane‐embedded sub‐proteomes remains challenging. Here, we experimentally characterized the inner membrane proteome (IMP) of the model organism E. coli BL21(DE3). We took advantage of the recent extensive re‐annotation of the theoretical E. coli IMP regarding the sub‐cellular localization of all its proteins. Using surface proteolysis of IMVs with variable chemical treatments followed by nanoLC‐MS/MS analysis, we experimentally identified ～45% of the expressed IMP in wild type E. coli BL21(DE3) with 242 proteins reported here for the first time. Using modified label‐free approaches we quantified 220 IM proteins. Finally, we compared protein levels between wild type cells and those over‐synthesizing the membrane‐embedded translocation channel SecYEG proteins. We propose that this proteomics pipeline will be generally applicable to the determination of IMP from other bacteria.     

含谷胱甘肽硫转移酶基因工程菌表达条件研究

将重组谷胱甘肽硫转移酶 (GST)大肠杆菌表达株BL2 1(DE3 )PGEX 作为研究对象 ,进行了生长及表达条件的优化研究 ,分析比较了不同培养条件对菌体生长的影响以及诱导剂的添加量对产物表达量的影响。